Insertion element IS3-based PCR method for subtyping Escherichia coli O157:H7.

An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified. While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates. IS3 PCR banding patterns were reproducible between amplifications and between subcultures. IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E. coli O157:H7 isolates.

Nitrate in baby foods. Adding to the nitrate mosaic.

OBJECTIVE: To identify commercial baby food varieties high in nitrate content using ion chromatography and compare the health risk associated with the consumption of high-nitrate water and high-nitrate commercial baby food. DESIGN: Ion chromatographic determination of nitrate concentration in a variety of commercial baby foods. SETTING: University Hygienic Laboratory, University of Iowa College of Medicine, Iowa City. PATIENTS: None. RESULTS: Commercial baby foods with nitrate levels higher than 45 ppm include mixed vegetables, bananas, carrots, garden vegetables, spinach, green beans, and beets. The amount of nitrate in one 113-g (4 oz) jar of beets, for example, is equivalent to the amount of nitrate in nearly 5.5 L of water at 45 ppm nitrate. CONCLUSIONS: A controlled clinical trial is needed to clarify how consuming high-nitrate foods correlates with methemoglobin levels in infants younger than 6 months.

Evaluation of multiple-marker screening for Down syndrome in a statewide population.

OBJECTIVE: Our purpose was to evaluate our experience with a statewide, multiple-marker Down syndrome screening program. STUDY DESIGN: The results of 18,712 screening tests performed from July 1, 1991, to Oct. 31, 1992, were reviewed. Amniocentesis and aneuploidy detection rates were compared with the experience of a previous year (1989-1990) in which material serum alpha-fetoprotein was used for detection of Down syndrome. RESULTS: Positive screening tests (Down syndrome risk > or = 1/190) occurred in 665 of 18,712 (3.5%) patients; 516 of 665 (78%) patients accepted amniocentesis. Fifteen aneuploidies were identified: 12 trisomy 21, one trisomy 18, one trisomy 13, and one 48,XXXY. The overall detection rate was one in 34 amniocenteses performed; for trisomy 21 it was one in 43. In a previous year in which maternal serum alpha-fetoprotein alone was used, 3.6% had positive screening tests (Down syndrome risk > or = 270); the detection rate for all aneuploidies was one in 57 amniocenteses, and for trisomy 21 it was one in 114. The expanded maternal serum alpha-fetoprotein test was well accepted by clinicians, with 36% of gravid state residents undergoing screening. CONCLUSION: The multiple marker test is a good screening tool and is superior to material serum alpha-fetoprotein alone.

HIV screening of newborns.

Iowa has participated in the national survey for the prevalence of HIV infection in childbearing women since July of 1989. As of February 1992, blinded testing for antibodies to HIV has been performed on blood spot specimens from 100,717 newborns. Of this number, 14 were confirmed as positive by Western blot. In the Iowa survey the prevalence of HIV infection in childbearing women was 0.14/1000 or 1/7000. This is similar to the prevalence that was observed for PKU in newborns during this time period. However, assuming only 30% of mothers transmit HIV to their babies, the predicted prevalence of HIV infection in Iowa newborns is 1/23,000. Certainly HIV disease is a public health concern with a frequency in Iowa mothers similar to that of other diseases screened for in the Iowa program. HIV meets the remaining WHO criteria for newborn screening, as well: the HIV screening test is simple and reliable and has a low incidence of false-positive and false-negative results; confirmatory testing, counseling, and medical care are available; the quality and length of life of affected individuals are improved by treatment; and data show that early diagnosis and treatment result in a cost advantage to society. The major obstacle to the addition of HIV testing to a newborn screening program is obtaining informed consent without jeopardizing program effectiveness.

Risk factors for human immunodeficiency virus (HIV) infections in homosexual men.

To clarify risk factors for infection with the human immunodeficiency virus (HIV) we selected at random 785 homosexual men who had participated in studies of hepatitis B in San Francisco in 1978-80 for a follow-up study of the acquired immunodeficiency syndrome. Although most had not been contacted in over five years, 492 (63 per cent) were located and enrolled. The 240 (67 per cent) who had developed antibodies to HIV, as measured by an enzyme-linked immunosorbent assay (ELISA), were compared with 119 who had remained seronegative. In multivariate analyses, receptive anal intercourse with ejaculation by nonsteady sexual partners, many sexual partners per month, and other indicators of high levels of sexual activity were highly associated with seroconversions. None of the sexual practices that we studied appeared to offer protection against HIV infection.

Detection of human T cell leukemia virus type I and human immunodeficiency virus in cultured lymphocytes of a Zairian man with AIDS.

We report the detection of human T cell leukemia virus type I (HTLV-I) and human immunodeficiency virus (HIV) in the cultured lymphocytes of a 45-year-old Zairian man with AIDS. HIV was successfully isolated and analyzed by SDS-PAGE and competition radioimmunoassay. However, by the culture techniques used, HTLV-I could not be separated from the HIV. Western blot analysis of the patient's serum showed the presence of both HTLV-I- and HIV-specific antibodies. The finding of this dual infection may explain reports that greater than or equal to 30% of patients with AIDS are positive for antibodies to HTLV-I.

Relationship between antibody to LAV/HTLV-III and the natural course of subclinical cellular immune dysfunction in homosexual men.

To assess the epidemiology and natural history of persistent generalized lymphadenopathy (PGL) and subclinical immunodeficiency in relation to serologic evidence of lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III) infection, 109 homosexual men with PGL, 47 homosexual men without lymphadenopathy who attended a sexually transmitted disease (STD) clinic, 25 homosexual male university students, and 26 heterosexual men who attended the STD clinic were studied. In 1982-1983 antibody to LAV/HTLV-III was present in 97%, 35%, 21%, and 4% of the four groups, respectively (P less than .001). Subclinical immunodeficiency was more closely associated with LAV/HTLV-III seropositivity than with lymphadenopathy. Cohorts of 78 homosexual subjects with PGL, 35 homosexual subjects from STD clinic, and 15 homosexual university students were followed for median periods of 13.5, 20, and 14.5 months, respectively. The seroconversion rate was 23% per year among seronegative subjects, and 4% of seropositive subjects developed overt acquired immunodeficiency syndrome (AIDS). Among seronegative subjects, there was significant improvement in T4:T8 ratios (P = .001), whereas most seropositive subjects continued to have subnormal total counts of T4 lymphocytes and low T4:T8 ratios. Some cases of subclinical cellular immunodeficiency apparently are unrelated to LAV/HTLV-III infection, and the presence of antibody to this virus is associated with an unfavorable immunologic prognosis.

Isolation of human immune deficiency virus from African AIDS patients and from persons without AIDS or IgG antibody to human immune deficiency virus.

We previously reported a high incidence of acquired immune deficiency syndrome (AIDS) in Kinshasa, Zaire, as well as a high frequency of antibody to human immunodeficiency virus (HIV), which includes HTLV-III and LAV viruses, in persons without AIDS. In this report we assessed the frequency of HIV virus infection in persons with and without clinical AIDS and the association of virus isolation to presence of antibody. We isolated HIV from 27 (77%) of 35 patients with AIDS, and 5 of 9 patients with AIDS-related complex (ARC). Virus was also isolated from plasma and cerebrospinal fluid of patients in the study. The presence of antibody was a reliable marker for virus infection in African patients with AIDS. HIV was isolated from 5 of 27 control patients without AIDS, 3 of whom had normal T helper to T suppressor ratios and normal numbers of T helper cells. Two of these patients had no detectable antibody to HIV by ELISA or Western blot methods. In a population, such as the general heterosexual population of Kinshasa, with frequent infection by HIV and with few clearly definable risk groups, screening for antibodies to HIV may not be sufficient to identify some virus infected persons.

PIP: This study represented the 1st attempt to isolate human immunodeficiency virus (HIV) from African acquired immunodeficiency syndrome (AIDS) patients and controls. HIV was isolated from 27 (77%) of 35 Zairians with AIDS and from 5 (55%) of 9 patients with AIDS-related complex (ARC). In addition, 5 (19%) of 27 controls admitted to Zaire's Mama Yemo Hospital for causes unrelated to AIDS were found to be positive for antibodies to HIV. Next, an effort was made to isolate the virus from 42 AIDS or ARC patients on whom data were already available on the results of an enzyme-linked immunosorbent assay (ELISA). HIV was isolated from 30 (81%) of 37 patients with positive ELISA tests and from none of the 5 patients with a negative assay. Among controls, antibodies were found in a higher proportion of patients with abnormal helper: suppressor ratios or a low absolute T helper cell count. On the other hand, these abnormalities were not found in 3 of the 5 control patients from whom HIV was isolated, including 2 without HIV antibody. This suggests that neither of these criterion are good indicators of virus infection. The isolation of HIV infection from 5 hospital controls with no clinical signs of infection suggests that either the rate of asymptomatic HIV virus infection is high in Zaire or that common tropical diseases such as malaria or tuberculosis may be associated with HIV infection. The frequency of HIV isolation from AIDS and ARC patients in this study is higher than that in earlier reports from non-Africans, but is comparable to current statistics from the US.

Human T-cell leukemia virus I provirus and antibodies in a captive gorilla with non-Hodgkin's lymphoma.

Antibodies reactive against human T-cell leukemia virus I (HTLV-I) were detected by indirect immunofluorescence assay using MT-2 as target cells, enzyme linked immunosorbent assay screen and competition assay, and Western blot analysis in three sera (one collected in 1979) from a captive gorilla which developed diffuse histiocytic lymphoma in 1983. The sera from four other healthy gorillas housed separately were HTLV-I antibody negative. All sera were negative for HTLV-III antibodies by enzyme linked immunosorbent assay. Southern blot analysis of DNA from lymphoma tissue after digestion with BamHI and using complete HTLV-I genome probe gave one 10-kilobase fragment and a characteristic 1.05-kilobase internal fragment detected in all known HTLV-I isolates. These results indicate that the gorilla was infected with HTLV-I or a closely related simian virus several years before the development of lymphoma.

Continuous production of a cytopathic human T-lymphotropic virus in a permissive neoplastic T-cell line.

We developed cloned populations from the commonly available, well-characterized cell line HUT-78. These cloned cells grow permanently after infection with isolates of human T-lymphotropic virus type III, also called lymphadenopathy virus (HTLV-III/LAV), from patients with acquired immune deficiency syndrome and related syndromes. In contrast, activated human T cells are lysed after HTLV-III/LAV infection. The infected cloned cells have been in culture continuously for 6 months and have produced high levels of extracellular reverse transcriptase (400,000 cpm/ml). This level is comparable to that of similarly infected normal human T cells. Three weeks after infection with HTLV-III/LAV, more than 90% of the cloned HUT-78 cells lysed; the remaining cells continued to grow. Approximately 80% of these cells expressed HTLV-III/LAV antigens by immunofluorescence. The extracellular virus of the chronically infected cell line was shown to be similar to other HTLV-III/LAV isolates by competition radioimmunoassay, by reactivity with human serum, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This HTLV-III/LAV-infected immortalized cell line enables the continuous production of large amounts of virus.

Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome.

The lymphadenopathy-associated virus (LAV) prototype strain of human T-lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow-up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact.

The natural history of infection with the lymphadenopathy-associated virus/human T-lymphotropic virus type III.

Over half of the persons infected with the lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III), the retrovirus that causes the acquired immunodeficiency syndrome (AIDS), become persistently infected with the virus. These "carriers" serve as the major reservoir of infection for others. Virus from their vascular and lymphatic spaces infects others through direct blood or mucous membrane exposure. After months to years, a high proportion of those infected will develop clinical manifestations of infection. For infected homosexual men, approximately 25% have developed AIDS-related conditions, mainly lymphadenopathy, and approximately 10% have developed AIDS. Because of the large number of infected persons in the United States, increasing rates of disease can be expected.

Transfusion-associated acquired immunodeficiency syndrome in the United States.

By Aug 15, 1985, one hundred ninety-four cases of possible transfusion-associated acquired immunodeficiency syndrome (AIDS) had been reported to the Centers for Disease Control. Cases received their transfusions in 30 states. Infants account for 10% of the cases, suggesting an increased susceptibility to developing AIDS. Investigations one to six years after the transfusions have identified high-risk donors to 47 cases. Of 47 high-risk donors tested, 40 had a reactive serology for human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) antibody, including five with no risk for AIDS by history. The HTLV-III/LAV was isolated from 23 of 26 seroreactive high-risk donors, most of whom remained asymptomatic. Blood components that transmitted HTLV-III/LAV included red cells, platelets, plasma, and whole blood. The time from transfusion to diagnosis of AIDS ranged from four to 84 months. The risk of developing AIDS after a blood transfusion has been low and will be lowered further by using both self-deferral and antibody screening.

Evidence against transmission of human T-lymphotropic virus/lymphadenopathy-associated virus (HTLV-III/LAV) in families of children with the acquired immunodeficiency syndrome.

Six children with the acquired immunodeficiency syndrome (AIDS) and 12 of their household contacts were investigated serologically for evidence of infection with human T-lymphotropic virus/lymphadenopathy-associated virus (HTLV-III/LAV), the presumed etiologic agent of AIDS. All six children had antibody against HTLV-III/LAV, as measured by enzyme-linked immunosorbent assay, in each specimen tested. Of the two mothers studied both were seropositive; one was diagnosed with and died from AIDS. Four of the remaining 10 household members were seropositive, including three adults in groups at high risk for the development of AIDS and one sibling who was younger than the child with AIDS. Among the seronegative household contacts were four foster mothers or grandmothers of the children with AIDS, three of whom had cared for the children since infancy. Household contact with children with AIDS may include persons in groups at high risk for AIDS who have been infected with HTLV-III/LAV. However, the negative findings in household contacts without risk factors for AIDS suggest that horizontal transmission of the virus within households by means other than sexual contact must be infrequent.